Gram-negative sepsis remains a major cause of mortality and morbidity in hospitalized patients, especially when complicated by acute kidney injury (AKI). The pathophysiology of AKI in sepsis continues to be poorly understood resulting in the persistent failure of clinical therapeutic trials. Recently, we identified a novel pathway of renal injury in sepsis involving direct interactions between filtered endotoxin and S1 proximal tubules. This endotoxin-S1 interaction resulted in severe peroxisomal damage and oxidative stress in downstream S2 and S3 segments. Remarkably, this pathway of injury had no requirement for competent immune cells. In this proposal, we continue our investigation of sepsis and AKI by examining the mechanisms of renal endotoxin preconditioning. The phenomenon of protective preconditioning is unique in that it represents a state of resistance to the deleterious effects of endotoxin and yet, a preserved ability to effectively contain and eliminate infections. Unraveling the pathways involved in endotoxin tolerance has great potential for identifying potential targets that can be used for the prevention and treatment of human sepsis. Historically, preconditioning has been investigated in immune cells and their isolated responses to repeated endotoxin exposure. Little is known about the mechanisms leading to tissue protection in whole organs such as the kidney. Based on strong preliminary data, the central hypothesis of this proposal is that macrophages are essential components of the protective pathways of preconditioning. This is a novel hypothesis because it depicts the macrophage as an active and beneficial participant in the tolerant phenotype. In specific aim 1, we will establish the essential role of macrophages and examine their cross-talk with renal tubules such as the S1 segment. In specific aim 2, we will determine the metabolic and transcriptomic changes imparted by protective macrophages on S1 tubules. We will also identify key metabolites that can be used directlt to treat sepsis. In specific aim 3, we will examine the potential of protective macrophages to treat sepsis in a cell transfer approach. We believe that the proposed studies, by increasing our understanding of endotoxin preconditioning, have great translational potential and will uncover a novel and global approach to the prevention and treatment of sepsis and sepsis-induced AKI.